CSF is a substance that acts on animal bone marrow cells so as to promote their differentiation and proliferation to macrophages or granulocytes. Several types of CSF have been reported. For example, Stanley, E. R. et al. reported that they purified from the urine of healthy adults a CSF that was composed of glycoprotein with a molecular weight of 45,000 and which exhibited a colony stimulating activity on mouse bone marrow cells but-not on human bone-marrow cells (Fed. Proce., 35, pp. 2272-2278, 1975). Burgess, A. W. et al. reported that a CSF that would be effective in humans was partially purified from human placenta (Blood, 49, 573-583, 1977, and ibid, 54, 614-627, 1979). Shah, R. G. et al. reported the partial purification of a similar CSF from monocytes in human peripheral blood and PHA-stimulated lymphocytes (Blood, 50, pp. 811, 1977). Fojo, S. S. et al. reported the partial purification of a similar CSF from the supernatant of a culture of human lungs (Biochemistry, 17, pp. 3109-3116, 1978). All of these CSFs are glycoproteins having molecular weights in the range of 25,000 to 41,000 and they act directly on non-adherent human bone marrow cells to form colonies of neutrophiles, macrophages and eosinophiles. However, because of limitations on the available sources, completely purified CSFs have not yet been obtained. In addition to these CSFs recovered from normal human tissues, some kinds of human tumor cells have recently been reported to have the capability of CSF production. For instance, Asano, S. et al reported the recovery of CSF from lung cancer cells transplanted into nude mice (Blood, 49, pp. 845-852 reported CSF production from a cell line of mandibular squamous cell carcinoma and thyroid gland cancer cells (Cancer Res., 38, pp. 3910-3917, 1978; JNCI, 69, pp. 1235-1243, 1982; and J. Cell Physiol., 110, pp. 43-49, 1982). Wu, M. C. et al. reported CSF recovery from a pancreas cancer cell line (J. Biol. Chem., 254, pp. 6226-6228, 1979; J. Clin. Invest., 65, pp. 772-775, 1980). Dipersio, J. F. et al. reported that they recovered CSF from a GCT Cell line established from patients with malignant histiocytoma (Blood, 51, pp. 1068, 1978; and Blood, 56, pp. 717-727, 1980). Golde, D. W. et al. reported their recovery of CSF from an MO cell line established from patients with hairy cell leukemia (Blood, 52, pp. 1068-1072, 1978; and Blood, 57, pp. 13-21, 1981). The CSFs, which are effective on human bone marrow cells are glycoproteins having molecular weights ranging from 27,000 to 34,000 and isoelectric points (pI) of 4.5-5.7. The CSF obtained from the supernatant of a culture of GCT cell line has been purified to a specific activity of 1.12.times.10.sup.6 U/mg. The specific acitivity of the CSF obtained from the supernatant of a culture of the MO cell line has been increased to 3.5.times.10.sup.6 U/mg. However, none of these CSFs have been purified completely. In addition, CSF that has the ability to specifically promote the differentiation and proliferation of human bone marrow cells to neutrophiles has not been reported to date.
The present inventors have suceeded in establishing a novel cell line from tumor cells in patients with oral cancer. The cell line had a great ability to produce CSF and exhibited highly proliferative capabilities. Named CHU-1, this cell line has been deposited with Collection Nationale de Cultures de Microorganismes, (C.N.C.M.) Pasteur Institute, France on July 11, 1984 under Deposit Number I-315.
The present inventors cultured this CHU-1 in vitro and successfully isolated from the supernatant of the culture a highly pure CSF which exhibited a human neutrophilic colony stimulating activity, and which had a molecular weight of about 18,000 (as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) and a specific activity of 3.94.times.10.sup.7 U/mg or higher. In one aspect, the present invention relates to a CSF having the following physicochemical properties which are not shown in literature and makes this CSF a novel substance.
(i) Molecular weight:
19,000.+-.1,000 as determined by sodium dodecylsulfatepolyacrylamide gel electrophoresis;
(ii) Isoelectric point:
Having at least one of the three isoelectric points,
A, B and C, shown in Table 1:
TABLE 1 ______________________________________ Isoelectric In the presence of In the absence of point (pI) 4 M urea urea ______________________________________ A 5.7 .+-. 0.1 5.5 .+-. 0.1 B 6.0 .+-. 0.1 5.8 .+-. 0.1 C 6.3 .+-. 0.1 6.1 .+-. 0.1 ______________________________________
(iii) UV absorption:
Maximum absorption at 280 nm and minimum absorption at 250 nm;
(iv) The following 21 amino acids are arranged from N-terminal: ##STR1##
In another aspect, the present invention relates to a method for obtaining a CSF, which comprises culturing a cell line having the ability to produce human G-CSF having the physicochemical properties shown above, subjecting the supernatant of the culture to steps (1) to (3) indicated below, and optionally subjecting the resulting fractions to either step (4) or (5):
(1) subjecting the supernatant of the culture to gel filtration using a gel having an effective fraction range of 5,000-70,000 daltons, and recovering fractions having the neutrophile-dominant colony stimulating activity (CSA);
(2) adsorbing the recovered fractions onto a carrier for reverse-phase high-performance liquid chromatography and performing elution by the density gradient technique with a mixture of water and an organic solvent so as to recover fractions having the neutrophile-dominant CSA;
(3) subjecting the so recovered fractions to high-performance molecular sieve chromatography so as to recover fractions having the neutrophile-dominant CSA;
(4) subjecting the so recovered fractions to isoelectric point electrophoresis so as to recover fractions having the neutrophile-dominant CSA; or
(5) subjecting the fractions recovered in step (3) to the step of removing sialic acid so as to recover fractions having the neutrophile-dominant CSA .